Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.
Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.
Pathogen infestation can lead to significant health issues in humans and animals. These health issues contribute to ever escalating human and animal healthcare expenditure. Even preventative measures require significant fiscal outlays.
Agricultural losses due to animal including poultry pathogens such as fungal pathogens, for example, are a major problem in the agricultural industry and each year millions of dollars are spent on the topical application by fungicides to curb these losses.
Although chemical and antibiotic pathogenicides including fungicides have been successful in human and veterinary medicine, the increasing use of these agents is providing selective pressure for emergence of resistant strains of pathogens. There is clearly a need to develop alternative mechanisms of controlling infestation by human and animal pathogens or to more efficiently manage existing agents.
Plants have evolved various systems to provide some natural protection against pathogen infestation. These innate immune systems comprise both constitutive or pre-formed and inducible components. Hithertofore, there has not been significant recognition of the potential application of the components of plant innate immune systems to non-plant hosts. Examples of these components are small, disulfide-rich proteins which play a large role in both the constitutive and inducible aspects of plant immunity. They can be categorized into families based on their cysteine arrangements and include the thionins, snakins, thaumatin-like proteins, hevein- and knottin-type proteins, lipid transfer proteins, hairpinins and cyclotides as well as defensins.
Plant defensins are small (45-54 amino acids), basic proteins with four to five disulfide bonds (Janssen et al. (2003) Biochemistry 42(27):8214-8222). They share a common disulfide bonding pattern and a common structural fold, in which a triple-stranded, antiparallel β-sheet is tethered to an α-helix by three disulfide bonds, forming a cysteine-stabilized αβ motif A fourth disulfide bond also joins the N- and C-termini leading to an extremely stable structure. A variety of functions has been attributed to defensins, including anti-bacterial activity, protein synthesis inhibition and α-amylase and protease inhibition (Colilla et al. (1990) FEBS Lett 270(1-2):191-194; Bloch and Richardson (1991) FEBS Lett 279(1):101-104).
The structure of defensins consists of seven ‘loops’, defined as the regions between cysteine residues. Loop 1 encompasses the first β-strand (1A) as well as most of the flexible region that connects this β-strand to the α-helix (1B) between the first two invariant cysteine residues. Loops 2, 3 and the beginning of 4 (4A) make up the α-helix, while the remaining loops (4B-7) make up β-strands 2 and 3 and the flexible region that connects them (β-hairpin region) (van der Weerden et al. (2013) Cell Mol Life Sci 70 (19): 3545-3570). This hairpin region of plant defensins forms a γ-core motif that is found in many anti-microbial peptides of diverse classes (Yount and Yeaman (2005) Protein Pept Lett 12(1):49-67).
Despite their conserved structure, plant defensins share very little sequence identity, with only the eight cysteine residues completely conserved. The cysteine residues are commonly referred to as “invariant cysteine residues”, as their presence and location are conserved amongst defensins. Based on sequence similarity, plant defensins can be categorized into different groups. Within each group, sequence homology is relatively high whereas inter-group amino acid similarity is low (van der Weerden et al. (2013) Cell Mol Life Sci 70 (19): 3545-3570).
Plant defensins can be divided into two major classes. Class I defensins consist of an endoplasmic reticulum (ER) signal sequence followed by a mature defensin domain. Class II defensins are produced as larger precursors with C-terminal pro-domains or pro-peptides (CTPPs) of about 33 amino acids. Most of the Class II defensins identified to date have been found in Solanaceous plant species.
Class II Solanaceous defensins are expressed in floral tissues. They include NaD1, which is expressed in high concentrations in the flowers of ornamental tobacco Nicotiana alata (Lay et al. (2003) Plant Physiol 131(3):1283-1293). The anti-fungal activity of this peptide involves binding to the cell wall, permeabilization of the plasma membrane and entry of the peptide into the cytoplasm of the hyphae (van der Weerden et al. (2008) J Biol Chem 283(21):14445-14452) and induction of reactive oxygen species (Hayes et al. (2014) Cell Mol Life Sci. February 2014, on line ISSN 1420-682X).
Class II Solanaceous defensins have variable degrees of activity against plant fungi. Some Class I defensins exhibit very low anti-fungal activity. Very little research has been conducted hithertofore on the effects of plant defensins on non-plant pathogens.
Defensins with highly divergent sequences act via different mechanism of actions. Permeabilization of the plasma membrane is a common feature that is observed for a number of defensins. However, the mechanism of permeabilization and its role in cell death differs between different defensins. Some defensins cause membrane permeabilization at high concentrations, but not at the concentration required for complete growth inhibition. In fact, the concentration of these proteins required to cause significant membrane permeabilization is around 20 times that required for growth inhibition. These proteins do cause slight membrane permeabilization at concentrations required for growth inhibition but only after long time periods (>150 mins). This is likely a result of fungal cell death that occurs after this time. The SYTOX green assay described in U.S. patent application Ser. No. 12/535,443 has been successfully used to assay permeabilization.
In contrast, to some other plant defensins, the plant defensin NaD1 causes significant membrane permeabilization at concentrations corresponding to the IC50. Permeabilization by NaD1 begins within 15 minutes and reaches its maximum after 80 minutes (van der Weerden et al. (2010) J Biol Chem 285(48):37513-37520). NaD1 also causes some membrane permeabilization at low concentrations that do not cause growth inhibition (van der Weerden et al. (2008) J Biol Chem 283(20:14445-14452). Difference in the permeabilization kinetics between defensins is likely due to differences in the mechanism of action of the proteins. Hence, there is a role in using permeabilization assays to select appropriate defensins.
There is a need to develop protocols to more effectively manage pathogen infection and infestation in humans and animals. The ability to more efficiently facilitate control of pathogens with reduced application of antibiotic agents or without the need for this application altogether will reduce the potential for resistant strains of pathogens to emerge. This is of particular concern, not only in hospital and healthcare facilities, but also within high density animal facilities.